WMIC 2015: WIMIN Scholar Award Winner 3

Normal Tissue Irradiation Promotes Tumor and Immune Cell Infiltration

Marjan Rafat, Stanford University

Innovation/Impact: Breast cancer recurrence rates remain high for triple-negative cases despite aggressive surgical, radiological, and chemotherapeutic intervention [1]. Recent studies suggest that recurrence may be facilitated by circulating tumor cell re-seeding of primary tumors [2]. The role of the tumor microenvironment in recurrence, however, is not well established.
Hypothesis/Objectives: Radiation has been shown to promote tumor cell migration in a preclinical breast cancer model [3]. We hypothesize that the irradiated tumor microenvironment influences tumor and immune cell behavior. In this study, we characterize the effects of normal tissue irradiation on tumor and immune cell invasion and migration to evaluate how tumor-stromal interactions modulate recurrence after therapy. This work represents the first step toward elucidating the contribution of stromal tissues in tumor and immune cell recruitment following radiotherapy.
Materials/Methods: Mouse embryonic fibroblasts (MEF) were irradiated to 20 Gy. Supernatant was collected after 2 or 7 d incubation and used as a chemoattractant in an in vitro transwell assay to investigate 4T1 murine or MDA-MB-231 human mammary carcinoma cell invasion. An orthotopic breast cancer model was also used to evaluate the effect of radiation on tumor cell migration to normal tissues. Luciferase-labeled 4T1 cells were used for in vivo and ex vivo bioluminescence imaging (BLI). Nude mice were inoculated with 4T1 cells in the mammary fat pad (MFP) and injected with PBS in the contralateral MFP. The contralateral normal MFP was irradiated to a dose of 20 Gy, and cell migration was monitored with BLI 10 days after irradiation. Immunohistochemistry (IHC) was performed to stain irradiated and control tissues for F4/80 to determine the extent of macrophage infiltration. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cells. Of particular interest were CD11b+F4/80+ and CD11b+GR1+ cell populations representing macrophages and myeloid-derived suppressor cells (MDSCs), respectively.
Results: Tumor cell migration to normal tissues both in vitro and in vivo was stimulated by radiation. 4T1 and MDA-MB-231 cells exhibited an increase in invasion (p<0.05) in vitro when exposed to irradiated MEF media. Ex vivo BLI analysis demonstrated that normal tissue irradiation attracted tumor cells to the MFP and surrounding tissues, including the peritoneum and muscle (p<0.01). IHC staining revealed an increase in macrophage infiltration in irradiated tissue sections. Flow cytometric analysis confirmed enhanced macrophage and MDSC recruitment in irradiated tissues.
Conclusions: Our study establishes that normal tissue radiation response may play a role in modulating tumor cell migration after radiation. The radiation-induced increase in macrophage and MDSC infiltration indicates that the immune system may contribute to tumor cell migration. These results suggest that the tumor stroma may facilitate tumor cell invasion and tumor regrowth following radiotherapy.
[1] Kaplan HG et al. The Breast J. 2009.
[2] Kim MY et al. Cell. 2009.
[3] Vilalta M et al. Cell Rep. 2014.

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